All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Neuromuscular junctions (NMJ) are highly specialized, morphologically distinct, and well-characterized cholinergic synapses [Hall and Sanes (1993) Cell 72 Suppl., 99-121]. Chronic impairments in NMJ activity induce neuromuscular disorders characterized by progressive deterioration of muscle structure and function. The molecular and cellular mechanisms leading from compromised NMJ activity to muscle wasting have not been elucidated.
One such disorder is myasthenia gravis (MG), caused by a defect in neuromuscular transmission mediated by auto-antibodies that severely reduce the number of functional post-synaptic muscle nicotinic acetylcholine receptors (nAChR) [Drachman D. G. (1994) N. Engl. J. Med. 330, 1797-1810; Vincent A. (1999) Curr. Opin. Neurol. 12, 545-551]. MG is characterized by fluctuating muscle weakness that may be transiently improved by inhibitors of acetylcholinesterase (AChE) [Penn A. S. and Rowland L. P. (1995) Myasthenia Gravis In: Meritt's Textbook of Neurology, 9th Edition, Williams and Wilkins, Baltimore, section XVII, 754-761]. The characteristic electrodiagnostic abnormality is a progressive, rapid, decline in the amplitude of compound muscle action potentials (CMAP) evoked by repetitive nerve stimulation at 3 or 5 Hz. To date, the standard treatment for MG includes immunosuppressive therapy combined with chronic administration of multiple daily doses of peripheral AChE inhibitors such as pyridostigmine (Mestinon™). While AChE inhibitors effectively restore muscle performance in MG patients, their effects are short-lived, calling for the development of additional effective treatment.
Antisense technology offers an attractive, gene-based alternative to conventional anti-cholinesterase therapeutics. Antisense technology exploits the rules of Watson-Crick base pairing to design short oligonucleotides, 15-25 residues in length, whose sequence is complementary to that of a target mRNA [Agrawal S. and Kandimalla E. R. (2000) Mol. Med. Today, 6, 72-81]. Stretches of double-stranded RNA, resulting from hybridization of the antisense oligonucleotide (ASON) with its target, activate RNAse H [Crooke S. T. (2000) Methods Enzymol. 313, 3-45] and promote specific degradation of the duplex mRNA. As antisense therapeutics target RNA rather than proteins, they offer the potential to design highly specific drugs with effective concentrations in the nanomolar range [Galyam N. et al. (2001) Antisense Nucleic Acid Drug Dev. 11, 51-57]. Phosphorothioated and 3′ terminally protected 2′-O-methyl antisense oligonucleotides targeted to mouse AChE mRNA were shown to be effective in blocking AChE expression in vitro in cultured human and rodent cells [Koenigsberger C. et al. (1997) J. Neurochem. 69, 1389-1397; WO 98/26062; Grisaru D. et al. (2001) Mol. Med. 7, 93-105], and in vivo in brain [Shohami E. et al. (2000) J. Mol. Med. 78, 278-236; Cohen et al. (2002) Molecular Psychiatry, in press], muscle [Lev-Lehman E. et al. (2000) J. Mol. Neurosci. 14, 93-105] and bone marrow [Grisarui et al. (2001) ibid.].
The inventors have recently observed that treatment with the irreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP) induces overexpression of an otherwise rare, non-synaptic alternative splicing variant of AChE, AChE-R, in brain [Kaufer D. et al. (1998) Nature, 393, 373-377] and intestine [Shapira M. et al. (2000) Hum. Mol. Genet. 9, 1273-1282]. Muscles from animals treated with DFP also overexpressed AChE-R, accompanied by exaggerated neurite branching, disorganized wasting fibers and proliferation of NMJs. Partially protected 2′-O-methyl antisense oligonucleotides targeted to mouse AChE mRNA suppressed feedback upregulation of AChE and ameliorated DFP-induced NMJ proliferation [Le-Lehman et al. (2000) ibid.]. These observations demonstrated that cholinergic stress elicits overexpression of AChE-R in muscle and that antisense oligonucleotides can suppress such AChE-R excess and prevent its deleterious outcome.
As mentioned above, the characteristic electrodiagnostic abnormality is a progressive, rapid decline in the amplitude of muscle action potentials evoked by repetitive nerve stimulation at 3 or 5 Hz. This myasthenic fatigue is caused by decrease in the number of AChR molecules available at the post-synaptic site. Inhibiting anti-AChR antibodies are present in 85% to 90% of patients [Vincent, A. (1999) id ibid].
Patients with MG, but not with congenital myasthenias due to other causes [Triggs et al. (1992) Muscle Nerve 15, 267-72], display a transient clinical response to AChE inhibitors such as edrophonium. The available anti-AChE drugs are the first line of treatment, but most patients require further help. This includes drastic measures, such as plasma exchange, thymectomy and immunosuppression. Unfortunately, all of the currently employed MG drug regimens are associated with deleterious long-term consequences. These include disturbance of neuromuscular transmission, exacerbation and induction of MG symptoms. Also, the otherwise safe use of common drugs such as anti-infectives, cardiovascular drugs, anticholinergics, anticonvulsants, antirheumatics and others has been reported to worsen the symptoms of MG patients [Wittbrodt (1997) Arch. Intern. Med., 157, 399-408].
While the neuromuscular malfunctioning associated with MG can be transiently alleviated by systemic chronic administration of carbamate acetylcholinesterase (AChE) inhibitors (e.g. pyridostigmine), the inventors have found that pyridostigmine induces a feedback response leading to excess AChE accumulation [Friedman et al. (1996) Nature Medicine 2, 1382-1385; Kaufer et al. (1998) id ibid; Meshorer, E. et al. (2002) Science 295, 508-12]. This suggested that the chronic use of such inhibitors would modify the cholinergic balance in the patients' neuromuscular system and Would require increased doses of these drugs; it also provided an explanation of the highly variable dose regimen employed in MG patients; and it called for the development of an alternative approach to suppress acetylcholine hydrolysis.
AChE-encoding RNA is subject to 3′ alternative splicing yielding mRNAs encoding a “synaptic” (S) isoform, containing exons 1-4 and 6, also designated E6 mRNA herein, an “erythrocytic” (E) isoform, containing exons 1-6, also designated E5 mRNA herein, and the “readthrough” AChE-R derived from the 3′-unspliced transcript, containing exons 1-6 and the pseudo-intron 14, also designated I4 mRNA herein.
Transgenic mice overexpressing human AChE-S in spinal cord motoneurons, but not in muscle, displayed progressive neuromotor impairments that were associated with changes in NMJ ultrastructure [Andres, C. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 8173-8178]. However, it was not clear whether the moderate extent of overexpressed AChE in muscle was itself sufficient to mediate this severe myopathology. In rodent brain, the inventors found previously that both traumatic stress and cholinesterase inhibitors induce dramatic calcium-dependent overexpression of AChE-R [Kaufer, et al. (1998) id ibid.], associated with neuronal hypersensitivity to both cholinergic agonists and antagonists [Meshorer et al. (2002) id ibid].
Chronic AChE excess was found to cause progressive neuromotor deterioration in transgenic mice and amphibian embryos [Ben Aziz-Aloya et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 2471-2475; Seidman et al. (1994) J. Neurochem. 62, 1670-1681; Seidman, et al. (1995) Mol. Cell. Biol. 15, 2993-3002; Andres, C. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 8173-8178; Sternfeld et al. (1998) J. Neurosci. 18, 1240-1249]. Also, myasthenic patients suffer acute crisis events, with a reported average annual incidence of 2.5% [Berrouschot et al. (1997) Crit. Care Med. 25, 1228-35] associated with respiratory failure reminiscent of anti-AChE intoxications.
In one approach, the prior art teaches that chemically protected RNA aptamers capable of blocking the autoantibodies to the nicotinic Acetylcholine Receptor (nAChR) may be developed and used to treat MG. This approach has several drawbacks in that the RNA aptamers do not have the amplification power characteristic of the RNAse-inducing antisense agents and in that it fails to address the problem of the feedback responses in MG.
The present inventors have previously found that antisense oligonucleotides against the common coding region of AChE are useful for suppressing AChE production [WO 98/26062]. This publication also teaches that antisense oligonucleotides against the human AChE are useful in the treatment of memory deficiencies as observed in transgenic mice that expressed human AChE in their brain. The observed effects (see Table 4-5 in WO 98/26062) are similar in their effect, yet considerably longer in the duration of their action than the prior art AChE inhibitor tacrine (see FIG. 9B in WO 98/26062).
In view of the above, it is desirable to further improve the treatment approaches for MG and other diseases involving impairment in neuromuscular transmission. The prior art treatment involving the use of AChE inhibitors is afflicted with undesirable side effects because of the induction of AChE and neuromuscular impairments by such inhibitors; and because it is subject to variable efficacy under altered mental state (stress).
WO01/36627 teaches that morphological and functional changes in the NMJ correlate with overexpression of a specific isoform of AChE mRNA, viz., the “readthrough” isoform containing the pseudo-intron I4 in the mature mRNA. Said PCT application also shows that antisense oligonucleotides directed to the common coding region of AChE may be used to specifically destroy AChE-R mRNA, and that AChE antisense agents are by far superior to conventional AChE enzyme inhibitor drugs in the treatment of neuromuscular disorders. The superiority of these antisense agents may be due to the fact that conventional enzyme inhibitors actively induce I4 AChE mRNA overexpression. According to the teachings of WO01/36627, this may lead to detrimental changes in the NMJ. This consequence of treatment may be entirely avoided by using the antisense agents of WO01/36627.
The Blood-Brain Barrier (BBB) maintains a homeostatic environment in the central nervous system (CNS). The capillaries that supply the blood to the brain have tight junctions which block the passage of most molecules through the capillary endothelial membranes. While the membranes do allow passage of lipid soluble materials, water soluble materials do not generally pass through the BBB. Mediated transport mechanisms exist to transport the water soluble glucose and essential amino acids through the BBB. Active support mechanisms remove molecules which become in excess, such as potassium, from the brain [for general review see Betz et al., Blood-Brain-Cerebrospinal Fluid Barriers, Chapter 32, in Basic Neurochemistry, 5th ed., Eds Siegel, Albers Agranoff, Molinoff, pp. 681-701; Goldstein and Betz (1986) Scientific American, September, pp. 74-83].
The BBB impedes the delivery of drugs to the CNS. Methods have been designed to deliver needed drugs such as direct delivery within the CNS by intrathecal delivery can be used with, for example, an Omaya reservoir. U.S. Pat. No. 5,455,044 provides for the use of a dispersion system for CNS delivery [for description of other CNS delivery mechanisms, see U.S. Pat. No. 5,558,852, Betz et al., ibid., and Goldstein and Betz, ibid.]. Tavitan et al. [Tavitan et al. (1998) Nat Med 4(4): 467-71] observed that 2′-O-methyl oligonucleotides are able to penetrate into the brain. Other systems make use of specially designed drugs that utilize the structure and function of the BBB itself to deliver the drugs, for example by designing lipid soluble drugs or by coupling to peptides that can penetrate the BBB.
It has been shown that stress affects the permeability of the BBB [Sharma H. S. et al. (1992) Prog. Brain Res. 91, 189-196; Ben-Nathan D. et al. (1991) Life. Sci. 489, 1493-1500]. Further, in mammals, acute stress elicits a rapid, transient increase in released acetylcholine with a corresponding phase of increased neuronal excitability [Imperato A. et al. (1991) Brain Res. 538, 111-117]. It has been previously observed by the present inventors that the AChE-R isoform and the I4 peptide of AChE can act as stress mimicking agents and rupture the BBB. These findings formed the basis for PCT application WO98/22132, the contents of which are fully incorporated herein by reference. WO98/22132 relates to compositions for facilitating the passage of compounds through the BBB, comprising the AChE-R splice variant and/or the peptide 14.
In search for an antisense oligonucleotide targeted against a domain of the human AChE, which may be particularly acceptable in human therapy, the inventors have now found, and this is an object of the present invention, that a synthetic antisense oligodeoxynucleotide having the nucleotide sequence: 5′-CTGCCACGTTCTCCTGCACC-3′, herein designated SEQ ID NO:1, is not only useful in selectively suppressing the production of the AChE-R isoform, but also possesses cross-species specificity, which enables its use in rodent animal models of various diseases and, moreover, remarkably appears to penetrate the BBB, and may thus be useful in treatment of diseases of the central nervous system, alone or in combination with other therapeutic agents. The finding that the novel antisense of the invention can penetrate the BBB was unexpected, particularly in view of the expectation that the BBB would be impermeable to large polar molecules.
The application of antisense technology to the treatment of nervous system disorders has, until recently, been considered to be limited by the lack of adequate systems for delivering oligonucleotides to the brain. Nevertheless, several attempts have been made to circumvent this difficulty [reviewed in Seidman S. et al. (1999) Antisense Nucl. Acid Drug Devel, 9, 333-340]. Access of chemical agents circulating in the blood to the interstitial spaces of the brain is restricted by the biomechanical barrier known as the BBB. The strong anionic character of the phosphodiester backbone makes oligonucleotides especially poor at crossing the BBB. In vivo pharmacokinetic studies have demonstrated that less than 0.01% of a systemically injected dose of a phosphorothioate antisense oligonucleotide may reach the brain, where its residence time may be as little as 60 min. A research solution to this problem in the laboratory is direct bypass of the BBB by intracranial injection of oligonucleotides. Using published stereotactic coordinates for both rats and mice, oligonucleotides can be delivered by single injections, by repeated administration through an implanted cannula, or by continuous infusion using an osmotic mini-pump such as Alzet (Alza, Palo Alto, Calif.). Oligonucleotides can either be delivered into the CSF or directly into the brain region of interest. In general, oligonucleotides are considered to remain relatively localized following intraparenchymal administration. Thus, a single injection of 24 μg of an antisense oligonucleotide targeted to the cAMP-response element (CREB) into rat amygdala was reported to diffuse only 0.72±0.04 μl around the injection site, exerting region-specific effects on conditioned taste aversion (CTA). Injection of the same oligonucleotide into the basal ganglia 2 mm above the amygdala had no effect on CTA. Similarly, specific effects on behavior were reported following the injection of antisense oligonucleotides against the stress-associated transcription factor c-fos into the medial frontal cortex (single administration; 10 μg), following delivery of oligonucleotides against the neurotransmitter-synthesizing enzyme glutamate decarboxylase into the ventromedial hypothalamus (single administration; 1 μg), and following 5 days continuous infusion of oligonucleotides targeted to mRNA encoding the cAMP-responsive transcription factor CREB into the locus coeruleus (20 μg/day). It was further reported that wide distribution of oligonucleotides in the brain (up to 443 μl around the site of injection after 48 hrs) could be achieved by direct, high-flow intraparenchymal microinfusion. In that case, the average tissue concentration of oligonucleotide was calculated to be between 3-15 μM—well within what is considered physiologically significant. Regarding uptake into neurons, it was shown that neurons in the striatum of rats preferentially take up oligonucleotides compared to glia. Despite the general retention of oligonucleotides around the injection site reported in that study, some signal was observed to be transported along projection pathways to distant sites. However, to be effective therapeutically, oligonucleotides should be prepared in a way that would enable their stability and free penetrance into the central nervous system following intravenous injection, or yet more preferably, following oral administration. Thus, the present invention is aimed at a novel, preferably nuclease protected antisense oligodeoxynucleotide targeted to the common coding domain of human AChE, which selectively suppresses the production of AChE-R, with rapid and long-lasting clinical improvements in muscle function, which possesses cross-species specificity and can penetrate the BBB and destroy AChE-R mRNA within central nervous system neurons.